Avian pathogenic Escherichia coli (APEC) and uropathogenic Escherichia coli (UPEC) : characterization and comparison

Introduction: Avian pathogenic E. coli (APEC) and uropathogenic E. coli (UPEC) are responsible for avian colibacillosis and human urinary tract infections, respectively. There are genetic similarities between the APEC and UPEC pathotypes, suggesting the APEC strains could be a potential reservoir of virulence and antimicrobial-resistance genes for the UPEC strains. This study aimed to characterize and compare APEC and UPEC strains regarding the phylogroup classification, pathogenicity and antimicrobial susceptibility. Methodology: A total of 238 APEC and 184 UPEC strains were selected and characterized. The strains were assayed for antimicrobial susceptibility and classified into phylogenetic groups using a multiplex-PCR protocol. In addition, the APEC strains had previously been classified according to their in vivo pathogenicity. Results: The results showed that both pathotypes had variation in their susceptibility to most of the antimicrobial agents evaluated, with few strains classified as multidrug resistant. The highest resistance rate for both pathotypes was to amoxicillin. Classifying the APEC and UPEC strains into phylogenetic groups determined that the most frequently frequencies were for groups D and B2, respectively. These results reflect the pathogenic potential of these strains, as all the UPEC strains were isolated from unhealthy patients, and most of the APEC strains were previously classified as pathogenic. Conclusions: The results indicate that distribution into phylogenetic groups provided, in part, similar classification to those of in vivo pathogenicity index, as it was possible to adequately differentiate most of the pathogenic and commensal or low-pathogenicity bacteria. However, no relationship could be found between the specific antimicrobial agents and pathogenicity or phylogenetic group for either pathotype

Perfil de lesões induzido por 293 amostras de Escherichia coli oriundas de frangos de corte, isoladas de quadros respiratórios, lesões cutâneas e cama de aviários, após inoculação em pintos de um dia

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Prevalence and distribution of pathogenic genes in Campylobacter jejuni isolated from poultry and human sources

Introduction: Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis. Despite its public health importance, the virulence factors and mechanisms underlying C. jejuni pathogenesis remain poorly understood and the relationships between these genes and the sources of the strains are not clear. We aimed to determine the virulence profiles of C. jejuni isolated from poultry and human cases of Campylobacteriosis. Methodology: A total of 50 strains of C. jejuni isolated from poultry and human cases of Campylobacteriosis were screened by polymerase chain reaction (PCR) for the presence of six pathogenic genes (flaA, iam, wlaN, cdtA, cdtB, cdtC). Results: A total of 40% (10/25) of the human isolates presented only one virulence marker. In contrast, 64% (16/25) of the poultry-derived strains showed four or five virulence markers. cdtA and flaA occurred more frequently in poultry-derived strains than in human strains. Ten different virulence profiles were observed among the human isolates and 11 among the poultry strains. Only four profiles were common to both sources: profiles 3 (flaA, cdtA, cdtB, and cdtC), 5 (cdtA and cdtB), 7 (flaA and cdtB), and 10 (iam, flaA, cdtA, cdtB, and cdtC). The human-derived strains had a higher Shannon diversity index (1.9396) and Simpson index (0.8367), indicating a more diversified population than found in poultry (1.7742 and 0.7333, respectively). Conclusions: We found variations in the genetic profiles of the circulating strains based on the isolation source and genes that are potentially pathogenic to humans were detected in poultry-derived strains

Avaliação da capacidade de formação de biofilme por cepas de Pasteurella multocida isoladas de casos de cólera aviária e de pulmões de suínos e sua relação com a patogenicidade

Pasteurella multocida is a Gram-negative bacillus that causes economic losses due to the development of respiratory diseases in several animal species. Among the mechanisms of virulence, the formation of biofilms is an important factor for bacterial survival in hostile environments. Studies of biofilm formation by P. multocida are needed because P. multocida is an important pathogen involved in respiratory infections. However, in contrast to other microorganisms, few studies of biofilm formation have examined P. multocida. Studies comparing the pathogenicity of microbial strains as a function of their biofilm production capacity are also rare. Consequently, the aim of this study was to evaluate the biofilm formation capacity of 94 P. multocida strains isolated from cases of fowl cholera and from swine lungs on polystyrene plates. The associations of the biofilm formation capacity with the pathogenicity index (PI) in vivo and with the presence of four genes (screened by PCR) of the tad locus (tadB, tadD, tadE and tadG), described as adhesion markers, were also determined. Strains from both animal origins were able to form biofilms. However, most of the specimens (52.13%) were classified as weak producers, and more than 40% of the strains of P. multocida (40.42%) did not produce biofilms There was no significant difference (p>0.05) in the degree of biofilm production between the two sources of isolation. Of the analyzed strains, 56.52% contained all four genes (tadB, tadD, tadE and tadG). The PI arithmetic mean of the strains classified as non-biofilm producers was significantly different (p0.05) with the production of biofilms and with the origin of a given strain. Finally, low virulence strains may suggest a higher biofilm formation capacity on polystyrene plates.Pasteurella multocida é um bacilo Gram negativo que ocasiona perdas econômicas, geralmente associadas a doenças respiratórias em diversas espécies animais. Entre os mecanismos de virulência existentes, a formação de biofilmes demonstra ser um importante fator para a proteção e para a sobrevivência bacteriana em ambientes hostis. Estudos relacionados à formação de biofilmes por P. multocida são necessários, uma vez que este é um importante patógeno envolvido em infecções respiratórias. Entretanto, ainda são poucos os estudos desenvolvidos nesta área, quando comparados com aqueles envolvendo outros microrganismos. Também são os raros os estudos que comparam a patogenicidade das cepas com a sua capacidade de produção de biofilme. Neste contexto, o objetivo deste estudo foi avaliar a capacidade de formação de biofilme em placas de poliestireno de 94 cepas de P. multocida isoladas de casos de cólera aviária e de pulmões de suínos, associando-se com o índice de patogenicidade (IP) in vivo e com a presença de quatro genes do locus tad (tadB, tadD, tadE e tadG), descritos como marcadores de adesão e pesquisados através de PCR. As cepas de ambas as origens foram capazes de formar biofilme. Contudo, a maioria dos exemplares (52,12%) foi classificada como fracamente produtora e mais de 40% das cepas de P. multocida (40,42%) não produziram biofilme Não foi observada diferença estatística (p>0,05) quanto ao grau de produção de biofilme entre as duas origens de isolamento. 56,52% das cepas analisadas apresentaram os quatro genes (tadB, tadD, tadE e tadG) concomitantemente. O IP médio das cepas classificadas como não produtoras de biofilme apresentou diferença estatística (p˂0,05) em relação ao IP das cepas moderadamente produtoras. Os exemplares classificados como fracamente produtores de biofilme diferiram significativamente (p˂0,05) do grupo de cepas moderadamente produtoras. Os resultados obtidos indicaram que, apesar de as cepas de P. multocida isoladas de casos de cólera aviária e do pulmão de suínos apresentarem capacidade de formar biofilme em superfícies de poliestireno, a adesão ocorreu geralmente de forma fraca. Os genes tadB, tadD, tadE e tadG, pertencentes ao locus tad, não apresentaram associação significativa com a produção de biofilme e nem com a origem de isolamento da cepa. Por fim, observou-se que as cepas de menor patogenicidade apresentaram uma maior capacidade de formação de biofilme em placas de poliestireno

Classification of Avian Pathogenic Escherichia coli by a Novel Pathogenicity Index Based on an Animal Model

Background: Avian Pathogenic Escherichia coli is the main agent of colibacillosis, a systemic disease that causes considerable economic losses to the poultry industry. In vivo experiments are used to measure the ability of E. coli to be pathogenic. Generally, these experiments have proposed different criteria for results interpretation and did not take into account the death time. The aim of this study was to propose a new methodology for the classification of E. coli pathogenicity by the establishment of a pathogenicity index based in the lethality, death time and the ability of the strain to cause colibacillosis lesions in challenged animals.Materials, Methods & Results: A total of 293 isolates of E. coli were randomly selected to this study. The strains were isolated from cellulitis lesions, broiler bedding material or respiratory diseases and were previously confirmed through biochemical profile. The bacterial isolates were kept frozen at -20°C. The strains were retrieved from stocks and cultured in brain-heart infusion broth overnight at 37°C to obtain a final concentration of 109 UFC/mL. A total of 2940 one-dayold chicks from commercial breeding hens were randomly assigned to groups containing 10 animals and each group was subcutaneously inoculated in the abdominal region with 0.1 mL of the standard inoculum solution containing each of the strains. A control group of 10 broilers were inoculated with 0.1 mL of brain-heart infusion broth by the same route. The chicks were kept for seven days. They were observed at intervals of 6, 12 and 24 h post-inoculation during the first days. From the second day on, the chicks were observed at intervals of 12 h. According to the death time and to the scores of each lesion (aerosaculitis, pericarditis, perihepatitis, peritonitis and cellulitis), a formula to determine the Individual Pathogenicity Index was established. A value of 10 was established as the maximum pathogenicity rate for an inoculated bird. From this rate, 5 points corresponded to scores for gross lesions present at necropsy. For each lesion present, it represents 1 point. The remaining 5 points corresponded to the death time. To obtain the death time value, an index of 1, corresponding to the maximum value assigned to a death on the first day, was divided by the number of days that the birds were evaluated, resulting in a value of 0.1428, which corresponded to a survival bonus factor. It was possible to classify E. coli strains into four pathogenicity groups according to the pathogenicity index: high pathogenicity (pathogenicity index ranging from 7 to 10), intermediate pathogenicity (pathogenicity index ranging from 4 to 6.99), low pathogenicity (pathogenicity index ranging from 1 to 3.99) and apathogenic (pathogenicity index ranging from 0 to 0.99). The analysis of the strains according to their origin revealed that isolates from broiler bedding material presented a lower pathogenicity index.Discussion: It is possible that the source of isolation implies in different results, depending on the criteria adopted. This data reinforces the importance of use a more accurate mathematical model to represents the biological phenomenon. In the study, all avian pathogenic Escherichia coli strains were classified based on a pathogenicity index and the concept of the death time represents an interesting parameter to measure the ability of the strain to promote acute and septicemic manifestation. The use of a support method for poultry veterinary diagnostic accompanying the fluctuation of the bacteria pathogenicity inside the farms may indicate a rational use of antimicrobial in poultry industry

Determining the best sectioning method and intestinal segment for morphometric analysis in broilers

Brazilian poultry production is very efficient and demands maximum broiler performance. Therefore, digestive system pathologies have a relevant role. Considering it is difficult to obtain consistent information on intestinal morphometric analysis, this study aimed at establishing essential and clear criteria for the collection of intestinal segments for morphometric analysis. Fifteen 13-d-old broilers were sacrificed and three intestinal segments were collected per bird. Two 3-cm long sections were obtained from each of the intestinal segments. Samples were collected open or closed. The closed samples were transversely, hemicylindrically, or longitudinally sectioned. Samples were processed and stained with hematoxylin and eosin. The number of microscopic fields in each section was counted. Villi presenting the base clearly embedded in the submucosa, no damage or folds, and simple columnar epithelium at the tip were considered adequate for measurements. These villi were counted in each sample. The results shows that hemicylindrical sections presented the highest number of observation fields, with an average of 9.76 fields. Jejunum samples were among the three highest average villi counts, with 18.23 in longitudinal sections and 15.61 in hemicylindrical sections. The results of the present study indicate that hemicylindrical sectioning and jejunal samples were, respectively, the best sectioning method and the best intestinal segment for the morphometric analysis of the intestines of broilers

A resistência a desinfetantes e a antimicrobianos não aumentou em um período de 10 anos (2006 a 2016) em Salmonella Heidelberg isoladas de granjas avícolas brasileiras

Salmonella is a major cause of foodborne illness worldwide, and poultry and its derived products are the most common food products associated with salmonellosis outbreaks. Some countries, including Brazil, have experienced an increased prevalence of Salmonella Heidelberg among their poultry flocks. Some isolates have also presented high resistance to antimicrobial agents and persist in the poultry farm environment. This study aimed to compare the susceptibility of S. Heidelberg strains isolated in 2006 with those isolated in 2016 against disinfectants and antimicrobial agents. The results showed that all the strains were highly susceptible to sodium hypochlorite, regardless of the conditions and year of isolation. Resistance to benzalkonium chloride varied according to the conditions applied, but not to the year of isolation. Increased antimicrobial resistance from 2006-2016 was observed only for tetracycline. The results suggest that the antimicrobial and disinfectant resistance of S. Heidelberg did not increase for ten years (2006-2016). However, further analysis should include a larger number of S. Heidelberg isolates from poultry origin and additional antimicrobial agents for more precise conclusions about the increasing in antimicrobial resistance in the last years.Salmonella é uma das principais causas das doenças transmitidas por alimento em todo o mundo, e a carne de frango e produtos derivados são os principais alimentos associados com surtos de salmonelose em humanos. Alguns países, incluindo o Brasil, têm observado um aumento da ocorrência de Salmonella Heidelberg nas suas granjas avícolas. Além disto, alguns isolados têm apresentado alta resistência aos antimicrobianos e têm persistido no ambiente de produção avícola. Neste contexto, o objetivo deste estudo foi comparar a susceptibilidade de cepas de S. Heidelberg isoladas em 2006 com aquelas isoladas em 2016 contra desinfetantes e agentes antimicrobianos. Os resultados demonstraram que as cepas foram altamente resistentes a hipoclorito de sódio, independentemente das condições e do ano de isolamento. A resistência ao cloreto de benzalcônio variou de acordo com as condições testadas, mas não com o ano de isolamento. Um aumento da resistência aos antimicrobianos de 2006 a 2016 foi observado apenas para tetraciclina. Os resultados sugerem que a resistência aos desinfetantes e aos antimicrobianos não aumentou em um período de dez anos (2006-2016). Entretanto, novas análises devem incluir um número maior de cepas de S. Heidelberg isoladas de fontes avícolas e outros agentes antimicrobianos para uma conclusão mais precisa sobre o aumento da resistência antimicrobiana nos últimos anos

Artificial neural networks on eggs production data management

Background: Eggs have acquired a greater importance as an inexpensive and high-quality protein. The Brazilian egg industry has been characterized by a constant production expansion in the last decade, increasing the number of housed animals and facilitating the spread of many diseases. In order to reduce the sanitary and financial risks, decisions regard¬ing the production and the health status of the flock must be made based on objective criteria. The use of Artificial Neural Networks (ANN) is a valuable tool to reduce the subjectivity of the analysis. In this context, the aim of this study was at validating the ANNs as viable tool to be employed in the prediction and management of commercial egg production flocks. Materials, Methods & Results: Data from 42 flocks of commercial layer hens from a poultry company were selected. The data refer to the period between 2010 and 2018 and it represents a total of 600,000 layers. Six parameters were selected as “output” data (number of dead birds per week, feed consumption, number of eggs, weekly weight, weekly egg produc¬tion and flock uniformity) and a total of 13 parameters were selected as “input” data (flock age, flock identification, total hens in the flock, weekly weight, flock uniformity, lineage, weekly mortality, absolute number of dead birds, eggs/hen, weekly egg production, feed consumption, flock location, creation phase). ANNs were elaborated by software programs NeuroShell Predictor and NeuroShell Classifier. The programs identified input variables for the assembly of the networks seeking the prediction of the variables called outgoing that are subsequently validated. This validation goes through the comparison between the predictions and the real data present in the database that was the basis for the work. Validation of each ANN is expressed by the specific statistical parameters multiple determination (R2) and Mean Squared Error (MSE). For instance, R2 above 0.70 expresses a good validation. ANN developed for the output variable “number of dead birds per week” presented R2= 0.9533 and MSE= 256.88. For “feed consumption”, the results were R2= 0.7382 and MSE= 274.56. For “number of eggs (eggs/hen)”, the results were R2= 0.9901 and MSE= 172.26. For “weekly weight”, R2= 0.9712 and MSE= 11154.41. For “weekly egg production”, R2= 0.8015 and MSE= 72.60. For “flock uniformity”, R2= -2.9955 and MSE= 431.82. Discussion: From the six ANN designed in this study, in five it was possible to validate the predictions by comparing predictions with the real data. In one output parameter (“flock uniformity”), it was not possible to have adequate validation due to insufficient data in our database. For “number of dead birds per week”, “feed consumption”, “weekly weight” and “uniformity”, the most important variable was “flock age” (27.5%, 52.5%, 55.2% and 37.9%, respectively). For “number of eggs (eggs/hen)”, “uniformity” (52.1%) was the most relevant variable for prediction. For “weekly egg production”, “flock age” and “number of eggs (eggs/hen)” were the most important zootechnical parameters, both with a relative contribution of 38.2%. The results showed that even with the use of a robust tool such as ANNs, it is necessary to have well-noted and clear information that expresses the reality of the flocks. In any case, the results presented allow us to state that ANNs are capable for the management of data generated in a commercial egg production facility. The process of evaluation of these data would be improved if ANNs were routinely used by the professionals linked to this activity

Identification of virulence-associated markers in Escherichia coli isolated from captive red-browed amazon parrot (Amazona rhodocorytha)

Due to the genetic similarity of pathogenic Escherichia coli isolated from birds and pathotypes of human origin, it is suggested that they have a common ancestor and may exchange virulence-associated genes. This study aimed to detect virulence-associated genes in E. coli strains isolated from the Red-browed Amazon parrot (Amazona rhodocorytha) kept at a conservation institute in Brazil. High genetic variability in virulence was observed, since 12 virulence profiles were found among 14 strains. The number of virulence-associated genes of single strains ranged from 5 to 22 out of 33 genes tested, and only one strain did not present any virulence genes. Regarding adhesion genes, most strains presented from two to five genes, and crlA (85.7%) and fimC (85.7%) were the most frequent. Frequencies were similar for invasion and iron acquisition genes. Variations among genes were observed for serum resistance and toxin-related genes. Some of the E. coli strains isolated from parrots presented virulence genes that are commonly associated with pathotypes of human origin, including newborn meningitis E. coli, uropathogenic E. coli, and sepsis-associated E. coli. It is noteworthy that some of these genes were present in the majority of the analyzed strains. Our results indicate that these strains detected in clinically healthy parrots can be potential reservoirs of several virulence-associated genes. These genes can be transmitted to other E. coli strains, including those that affect humans. These E. coli strains present a high pathogenic potential of virulence-associated genes in extraintestinal pathogenic E. coli strains

Aflatoxicose em criação de marrecos no sul do Brasil

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